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Identification of RNAs associated with telomeres by enChIP-RNA-Seq

ABSTRACT: Accumulating evidence suggests important roles of RNAs interacting with genomic regions in the regulation of genome functions including X chromosome inactivation and gene expression. However, no method to identify RNAs interacting with a given genomic region in a non-biased manner has been reported. Here, we used engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) combined with the RNA-Seq analysis (enChIP-RNA-Seq) to perform non-biased search of RNAs interacting with telomeres. In enChIP-RNA-Seq, the target genomic regions are captured with an engineered DNA-binding molecule such as a TAL protein. Subsequently, RNAs are purified and subjected to the RNA-Seq analysis. The detected RNAs contained known telomere-binding RNAs including telomerase RNA and Cajal body-specific RNAs. In addition, we detected many novel telomere-binding RNAs. We confirmed binding of candidate RNAs to telomeres by the enChIP-RT-PCR analysis. Identified novel telomere-binding RNAs may play important roles in telomere functions. In addition, our results suggest that enChIP-RNA-Seq analysis would be useful for identification of RNAs interacting with specific genomic regions.

ORGANISM(S): Mus musculus

PROVIDER: GSE60425 | GEO | 2015/04/16

REPOSITORIES: GEO

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Project description:Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1/Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Î mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of the FPC in regulation of telomere stability in cancer cells. Genome-wide distributions of Rad52 in wild type and in swi1 deletion in fission yeast The'SP1173_WT ChIP-seq' is an input sample (non-tagged data).

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Identification of RNAs associated with telomeres by enChIP-RNA-Seq

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